A. pleuropneumoniae serotype 2, isolated in Korea, was used for the genes of apxIIA and apxIIIA [12]. The apxIA gene was obtained from A. pleuropneumoniae serotype 5 isolated from a Korean pig with pleuropneumonia. The bacteria were cultured in Luria-Bertani (LB) broth (Difco, USA) with 0.01% β-nicotinamide adenine dinucleotide for 24 h at 37°C.

Protein sequence information regarding apxIA (AF363361), apxIIA (AF363362), and apxIIIA (AF363363) was retrieved from the National Center for Biotechnology Information database. Homology and conserved domains were investigated using multiple sequence alignment of the ClustalW2 program (http://www.ebi.ac.uk/Tools/clustalw) to identify specific gene regions among toxins.

The gene sequence of each toxin was divided into four regions, the N-terminal (Nt), middle part 1 (M1), middle part 2 (M2), and C-terminal (Ct), for investigation of specific gene regions. Specific gene regions showing low levels of homology were determined based on the results of alignment of the four gene regions of each toxin. The specific gene regions identified for each toxin (partial Apx toxins) were amplified by polymerase chain reaction (PCR) using the primers described in Table 1. Total genomic DNA extracted from A. pleuropneumoniae serotype 2 and 5 was used for PCR templates (GenElute; Sigma-Aldrich, USA). The PCR products were electrophoresed in 1.0% agarose gel and then purified with a gel extraction kit (QIAquick; Qiagen, Germany). Amplified gene regions for each toxin were cloned with the Chapion pET100 vector (Invitrogen, USA) into Top10 chemical-component Escherichia coli cells (Invitrogen) according to the manufacturer's protocols. To confirm gene insertion, DNA sequencing was conducted using an automated DNA sequencer (ABI377L; Applied Biosystems, USA). Following confirmation of gene insertion by DNA sequencing, the cloned partial Apx toxins were transformed into component E. coli M15 cells for expression.

E. coli M15 cells were grown in LB broth with ampicillin (100 µg/mL) at 37°C. When the culture reached an optical density (OD) of 0.6 nm, isopropyl β-D-1-thiogalactopyranoside (1 mM; Duchefa Biochemie, the Netherlands) was added and then cultured continuously for 4 h. The harvested cells were subsequently re-suspended in lysis buffer (20 mM Tris-hydrogen chloride, 500 mM sodium chloride, 8 M urea, 40 mM imidazole, pH 7.0). Nickel-nitrilotriacetic acid chelate affinity chromatography (GE Healthcare, UK) was conducted based on the manufacturer's protocols, after which the bound proteins were eluted with elution buffer (20 mM Tris-hydrogen chloride, 500 mM sodium chloride, 8 M urea, 500 mM imidazole, pH 7.0). The concentration of the purified recombinant proteins was then determined using a BCA Protein Assay kit (Pierce, USA)

The purified partial-Apx toxins were separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue R-250. For Western blot analysis, the separated proteins from the SDS-PAGE were electrotransferred onto an iBlot Gel Transfer Stacks nitrocellulose membrane (Invitrogen), which was then immunoblotted with sera (1:1,000) of guinea pigs vaccinated with subunit vaccines containing ApxI, ApxII, and ApxIII toxoids, respectively. An alkaline phosphatase-conjugated rabbit anti-pig IgG (H+L) at 1:2,000 dilution was used as the secondary antibody and the specific reaction was detected using an AP-conjugated substrate kit (BioRad, USA). The predicted size of the purified partial-Apx toxins matched the results of the SDS-PAGE and Western blot analyses based on amino acids constitution.

The full-length ApxIA, ApxIIA, and ApxIIIA are recombinant proteins and major components of the A. pleuropneumoniae vaccine newly developed by Choong Ang Vaccine Laboratories Company (Daejeon, Korea). Guinea pig vaccination was carried out three times with these full-length Apx toxoids by intramuscular injection of 200 µg of each toxin at two-week intervals, respectively. Sampling of sera was performed two days after the last administration. All experiments using guinea pigs were conducted in accordance with the procedures permitted by the Institutional Animal Care and Use Committees of Choong Ang Vaccine Laboratories Company (No. 160129-01).

The assays were developed through checkerboard titration of 96-well microplates utilizing representative positive and negative sera. Sensitization of partial recombinant ApxIA, ApxIIA, and ApxIIIA antigens was performed using a two-fold serial dilution of each antigen at levels ranging from 3.125 ng/well to 200 ng/well. Antigens of partial-recombinant Apx toxins were coated onto 96-well microplates with 50 mM coating buffer (15 mM sodium carbonate and 35 mM sodium bicarbonate, pH 9.6), then incubated at 4°C overnight. Next, microplates were washed with a solution of phosphate-buffered saline containing 0.05% Tween 20 (PBST), after which they were blocked at room temperature for 2 h using 3% bovine serum albumin fraction V (BSA; Fisher Scientific, USA) in PBST. Sera from vaccinated guinea pigs with each full-length Apx toxin and serum from a nonvaccinated guinea pig were used as positive and negative controls, respectively. The microplates were incubated at room temperature for 2 h with 100 µL of two-fold serial diluted sera, after which they were incubated with 100 µL of 1:10,000 diluted horseradish peroxidase-conjugated goat anti-guinea pig IgG (H+L) (BioRad) at room temperature for 1 h. Sera and antibody used in ELISA were diluted with 1% BSA in PBST. Color development was performed with 3,3′,5,5′-tetramethylbenzidine liquid substrate (Sigma-Aldrich) at room temperature for 10 min. The color development was stopped with 1 N sulfuric acid, after which the microplates were read at 450 nm. Based on the optimal ELISA conditions confirmed by checkerboard titration, ApxI, ApxII, and ApxIII-specific ELISA conditions were established under the same conditions of the checkerboard titration; however, 5% (for blocking) and 3% (for dilution of sera and antibody) skim milk in PBST were used.

Data are presented as the means ± standard deviation. Statistical significance was determined by Student's t-tests using SPSS version 23.0 (SPSS, USA). A p < 0.05 was considered to indicate statistical significance.

All of the Apx toxins showed > 40% homology with each other (Table 2). The results of homology analysis between regions within each toxin (Nt, M1, M2, and Ct) are presented in Table 3. The M2 region was considered to be inadequate for the specific gene region based on high levels of homology. Based on the low percent identity observed upon homology analysis, specific gene regions were determined for ELISA antigen candidate peptides as follows: Nt and Ct regions in ApxIA; Nt, M2, and Ct regions in ApxIIA; Nt, M2, and Ct regions in ApxIIIA. The determined sequence regions are shown in yellow in Fig. 1.

Based on the results of homology analysis, all selected specific gene regions except ApxIIA Ct were amplified using the primers shown in Table 1 and then cloned for production of recombinant proteins. As shown in Fig. 2, the molecular masses of the recombinant proteins were in concordance with the predicted molecular weights (ApxIA Nt, 19.3 kDa; ApxIA Ct, 24.2 kDa; ApxIIA Nt, 19.1 kDa; ApxIIA M2, 21.3 kDa; ApxIIIA Nt, 20.1 kDa; ApxIIIA M2, 22 kDa; ApxIIIA Ct, 25.9 kDa). Upon Western blot analysis, the recombinant proteins were found to be responsive to the positive control of guinea pig sera, with cross reactions observed between the purified partial toxins upon homology analysis as expected.

Checkerboard titration was performed to establish the optimal ELISA conditions making a clear distinction between positive and negative sera. The optimal concentrations of recombinant partial toxins used to coat the microplates were as follows: 100 ng for ApxIA Nt, 200 ng for ApxIA Ct, 200 ng for ApxIIA Nt, 50 ng for ApxIIA M2, 200 ng for ApxIIIA Nt, 200 ng for ApxIIIA M2, and 100 ng for ApxIIIA Ct (Supplementary Figs. 1, 2, 3). To establish ELISA conditions for measuring ApxIA, ApxII, and ApxIIIA-specific antibody levels using the recombinant partial toxins, purified proteins of ApxIA Ct, ApxIIA Nt, and ApxIIIA Nt were selected as ELISA antigens considering the cross-reactivity (Fig. 3). Based on the checkerboard results and selected antigens, a 1:320 dilution ratio of guinea pig sera was found to show the most precise results. As shown in Fig. 3, ApxIA Ct (200 ng/well) had higher reactivity with ApxIA positive guinea pig sera than ApxIIA Nt (200 ng/well) and ApxIIIA Nt (200 ng/well). ApxIIA Nt and ApxIIIA Nt also showed greater responses to each positive serum than the other two antigens.